Download SOL-GEL NANOMATERIALS WITH ALGAL

Articles
MB
SOL-GEL NANOMATERIALS WITH ALGAL HETEROPOLYSACCHARIDE
FOR IMMOBILIZATION OF MICROBIAL CELLS, PRODUCING
α-GALACTOSIDASE AND NITRILASE
P. Djambaski1, P. Aleksieva2, E. Emanuilova2, G. Chernev1, D. Spasova2, L. Nacheva2, L. Kabaivanova2, I.M. Miranda Salvado3,
B. Samuneva1†
University of Chemical Technology and Metallurgy, Department of Silicate Technology, Sofia, Bulgaria1
Bulgarian Academy of Sciences, Institute of Microbiology, Sofia, Bulgaria2
University of Aveiro, Department of Ceramic and Glass Technology, CICECO, Aveiro, Portugal3
Correspondence to: Lilyana Nacheva or Georgi Chernev
E-mail: [email protected]; [email protected]
ABSTRACT
The main purpose of the present work is the sol-gel synthesis and structure of the hybrid nanomaterials as matrices for two types
of cells, producing hydrolytic enzymes. The effect of different percent of algal polysaccharide included in them on the hydrolytic
activity of fungal and bacterial cells was investigated.
The hybrid sol-gel nanomaterials were synthesized from tetraethylortosilicate (TEOS) as a silicon precursor and
heteropolysaccharide (AHPS) from the red microalga Dixonella grisea as an organic part. The structure of these matrices
was investigated using different methods: FT-IR, XRD, BET-Analysis, EDS, SEM and AFM. The sol-gel hybrids were used
for the immobilization of fungal (Humicola lutea) and bacterial (Bacillus sp.) cells, producing α-galactosidase and nitrilase,
respectively. It was established the effect of the quantity of the heteropolysaccharide in the matrices on the activity of these
hydrolytic enzymes. Using 20% AHPS in the hybrid nanomaterials the α-galactosidase yield exceeded over two-fold the enzyme
titre of the free cells in the third cycle of repeated batch shake flask cultivation. These results correlated with a dense growth of
immobilized mycelium observed with scanning electron microscopy (SEM). The increase of the percentage of organic part in the
sol-gel matrix up to 20% led to an increase in the nitrilase activity. The addition of 40% AHPS did not significantly affect the
decrease of the nitrile biodegradation.
Keywords: Sol-gel nanomaterial, microbial immobilization,
hydrolytic enzymes
Introduction
The sol-gel process consists of the hydrolysis and
condensation of organometallic compound precursors such as
tetramethoxysilane (TMOS) [Si(OCH3)4] and tetraethoxysilane
[Si(OC2H5)4]. A silicate synthesized by this process is a porous
material having pores large enough to allow diffusion. The
pore size can be easily controlled during the sol–gel process
by changing several conditions such as pH, aging time, and
mixing procedure. In addition, the sol–gel process can be
performed at room temperature. These properties are useful
for immobilizing biomolecules while retaining their biological
activities and allowing bioreactions to proceed inside the
matrix. Therefore, such bioencapsulations in sol–gel matrices
have been widely studied in recent years (11, 18, 20).
Microbial α-galactosidase (E.C.3.2.1.22), catalyzing
the hydrolysis of simple and complex oligosaccharides
and polysaccharides with α-1,6 linked terminal galactosyl
groups, is of particular interest in view of its applications.
This enzyme is used in food processing, the sugar industry,
medicine, enzymatic synthesis and structural analysis (13,
15). Most investigated α-galactosidases are produced by free
cells of filamentous fungi, belonging to the genera Aspergillus,
1270
Penicillium and Humicola (1, 12, 14). The immobilization
of the enzyme, that can be applied for removal of flatulenceinducing sugars in soymilk was reported (16). The existing data
about α-galactosidase biosynthesis by immobilized fungal cells
in inorganic-organic hybrid sol-gel materials is insufficient (6).
Microbial nitrilases (E.C. 3.5.5.1) are versatile nitrilemetabolizing enzymes used as an alternative of nitrile
hydrolysis instead of strong acids or base catalysts. They can
be involved in the production of acrylamide (4) and for the
synthesis of nicotinamide, nicotinic acid from cyanopiridin
or to carry out selective transformations (23). Most nitriles
are highly toxic and their microbial degradation has been
considered as an efficient way for detoxification of industrially
polluted waters and soils. Biochemical conversions, including
biodegradation that can be carried out by free or immobilized
cells, especially at high temperatures have been of great
interest (9, 17). The immobilized biocatalysts possess many
advantages such as improved stability and reusability.
In our previous works we have compared pure silica matrices
with inorganic-organic hybrid matrices obtained by using
different inorganic precursors: tetraethylortosilicate (TEOS),
tetramethylortosilicate
(TMOS),
ethyltrimethoxysilane
(ETMS), methyltriethoxysilane (MTES) and an organic
compound such as: polyethylene oxide (PEO), polyacrylamide
gel (PAAG), Ca alginate, gelatin and others (5, 10, 19).
Biotechnol. & Biotechnol. Eq. 23/2009/2
In this paper the research results on sol-gel synthesis and
structure of hybrid nanocomposites containing TEOS as a
silicon precursor and algal heteropolysaccharide (AHPS) as an
organic part are described and discussed. The influence of the
percentage (20 and 40 wt. %) of AHPS in the hybrid sol-gel
matrix used for the encapsulation of two types of cells on the
α-galactosidase production during repeated batch shake-flask
cultivation of the fungus Humicola lutea 120-5 and the value
of nitrilase activity of free and immobilized Bacillus sp. UG5B cells was investigated.
For the scanning electron microscopy (SEM) observations
the samples with immobilized H. lutea cells were fixed for
2 h with 2% (w/v) glutaraldehyde (3) at room temperature.
After washing with distilled water samples were dehydrated
in ethanol series ranging from 30 to 100% (v/v). After airdrying, specimens were coated with 120 - 130 Å gold in argon
atmosphere using an Edwards apparatus (model S 150A). The
SEM observations were made on a Philips SEM 515 at 20 kV
accelerating voltage with a 5 - 6 nm electron beam.
Materials and Methods
The results from the XRD-analysis show that all the studied
hybrids are in amorphous state (Fig. 1). It is visible that
with introducing of organic component into synthesized
nanobiomaterials, intensity of peaks decreases.
The samples have been prepared at room temperature as films.
Silicon precursor tetraethylortosilicate (TEOS) purchased by
“Merck” has been used. A poly-step sol-gel procedure was
used at strictly controlled conditions in order to obtain the
desired nanostructred materials. In all cases the ratio precursor
/H2O/ 0.1N HCl was kept 1/1/0.01. The acid was introduced to
increase hydrolysis rate (pH~1.5).
The algal heteropolysaccharide used in this study was
isolated from the red microalga Dixonella grisea (former Rh.
reticulata) UTEX LB 2320 (algal collection of the Department
of Botany, University of Austin, Texas, USA).
The fungal strain Humicola lutea 120-5 and the bacterial
strain Bacillus sp. UG-5B (National Bank for Industrial
Microorganisms and Cell Cultures, Bulgaria, No 391 and No
8021, respectively) were used in this study.
Immobilization of the fungal cells was carried out using
spore suspension (5 ml) with a density 1010 spores per ml
from one test tube. The cultivation of immobilized cells was
performed in 500 ml Erlenmeyer flasks with 50 ml soy meal
extract as a nutrient medium on a rotary shaker during 144 h
at 30°C (2). α-Galactosidase was assayed using the method of
Dey et al. (7). One unit (U) of α-galactosidase activity was
defined as the amount of enzyme which liberates 1 μmol of
p-nitrophenol per minute at pH 5.5 and 50°C.
Bacterial cell suspension with a density of 35 mg.ml-1 cells
was used in the immobilization process. It was prepared by
centrifugation of the culture material and re-suspension in a
buffer solution (0.06M, pH 7.2 at 20°C). The nitrilase action
leads to the direct hydrolysis of benzonitrile to benzoic acid and
ammonia and was assayed by measuring the ammonia released
according to the method of Fawcett and Scott (8). One enzyme
unit (U) is defined as the amount of enzyme, producing 1mmol
ammonia min-1 at pH 7.2, 45°C and 20 mM substrate.
The enzyme activities were evaluated from two repeated
experiments using tree parallel samples. The deviation of
replicates was less than 3%.
For studying the structure of synthesized hybrids the
following methods have been used: FT-IR (IR- MATSON
7000–FTIR spectrometer), XRD (X-ray PW1730/10
diffractometer), BET-Analysis (Gemini 2370 V5.00) and AFM
(NanoScope Tapping ModeTM).
Biotechnol. & Biotechnol. Eq. 23/2009/2
Results and Discussion
Fig. 1. XRD patterns of silica hybrids containing TEOS and 20 wt. % algal
polysaccharide
Fig. 2. FT-IR spectra of silica hybrids containing TEOS and 20 wt. % algal
polysaccharide
The FT-IR spectra (Fig. 2) of synthesized inorganicorganic materials show that in all samples, bands at 1080 cm-1,
1271
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
l)
Fig. 3. AFM three and two dimentional image and height distribution profile of surface roughness of hybrid material containing TEOS and 5 (a, b, c), 10 (d, e,
f), 15 (g, h, i) and 20 (j, k, l) wt. % algal polysaccharide.
790 cm-1 and 480 cm-1 are observed. They are assigned to νas,
νs and δ of Si-O-Si vibrations, but at the same time the band
at 1080 cm-1 can be related to the presence of Si-O-C, C-O-C
and Si-C bonds. The band at 960 cm-1 is due to a stretching
Si-OH vibration. The band at 1439 cm-1 is assigned to C-O-H
vibrations. The characteristic bands at around 3450 cm-1 and at
1620 cm-1 assigned to H-O-H vibration can also be detected.
The presence of a hybrid nanostructure with well-defined
nanounits and their aggregates, formed by self-organizing
1272
processes, is clearly observed by AFM studies. The size
of nanoparticles is from 3 to 7 nm (Fig. 3 a, d, g, j) show
the height distribution profiles of surfaces roughness. The
histograms of the surface height distribution profiles, obtained
from AFM images, show that the inorganic-organic hybrid
sample has surface with irregularities of quite small height
(Fig. 3 c, f, i, l).
The surface morphology and structure of nanobuilding
blocks in each synthesized hybrid is different and depends
Biotechnol. & Biotechnol. Eq. 23/2009/2
TABLE 1
Relative nitrilase activity of free and immobilized cells
Substrate for degradation
m-tolunitrile
m-tolunitrile
m-tolunitrile
p-tolunitrile
p-tolunitrile
p-tolunitrile
o-tolunitrile
o-tolunitrile
o-tolunitrile
Relative nitrilase activity, %
Percent of organic part
(AHPS) in the hybrid matrix Free cells
5%
30
20%
30
40%
30
5%
20
20%
20
40%
20
5%
14
20%
14
40%
14
on its chemical composition. In the sample the nanoparticles
are well distributed in the entire hybrid matrix with a lower
degree of aggregation, while the sample prepared with TEOS
show that the nanoparticles are self-organized and distributed
as clusters in the matrix. Although all being amorphous, quite
different self-organized structures can be observed in these
hybrids.
Relative activities (%)
20%
40%
250
200
150
100
50
Encapsulated cells
70
80
74
34
48
48
20
32
29
maximal productivity clearly manifested the relation between
the cell morphology and enzyme yield. After five fermentation
cycles or 30 days the α-galactosidase activity decreased (Fig.
4) and the immobilized mycelium became distorted and partly
autolyzed. Deformed wrinkled hyphae, empty of cell content
and spore formation were observed (Fig. 6). Good operation
stability was also proved. As can be seen (Fig. 4) during 3
incubations (18 days) the α-galactosidase activity exceeded the
enzyme level of free cells. Similar results were obtained using
H. lutea immobilized in hybrid sol-gel consisting of TEOS and
a mixture of polyethyleneglycol (PEG) and polyvinylalcohol
(PVA) (22). When the percent of AHPS in the hybrid matrix
was increased to 40% in the first cycle the enzyme activity
was the same with that of the free cells, but in the consecutive
re-uses the α-galactosidase titer started to decrease. Probably
the lower enzyme yield in the samples with higher organic
concentration is a result of a decrease of stability of the solgel particles in the conditions of repeated-batch shake flask
cultivation, where every cycle was 6 days long.
0
0
1
2
3
4
5
Batch number
Fig. 4. α-Galactosidase production by H. lutea cells, immobilized in the
matrix with 20% AHPS (-♦-) and 40% (-▲-) during repeated batch shake flask
cultivation
α-Galactosidase production by fungal cells immobilized
in the hybrid matrices, containing 20% and 40% AHPS was
followed. On Fig. 4 the relative activities during repeated
batch shake-flask cultivation were presented. Precultivation
time (the period for the fungal development from the entrapped
spores) as well as each batch cycle continued 144 h. Better
results were obtained using the matrix with 20% AHPS, when
in the third cycle the enzyme titer was higher (51U/flask or
232%) as compared to the free cell fermentation (22U or
100%). The high α-galactosidase activity correlated with dense
growth of immobilized mycelium observed with SEM (Fig. 5).
The viable elongated and branched hyphae in the third cycle of
Biotechnol. & Biotechnol. Eq. 23/2009/2
Fig. 5. SEM of H. lutea immobilized in hybrid matrix containing 20% AHPS,
showing a dense mycelial growth in the third cycle of semicontinuous shake
flask process. Bar= 10µm
1273
Sharp increase in the enzyme activity of the encapsulated
bacterial cells was registered compared to these of the free cells
(Table 1). As a control (100% enzyme activity) corresponds to
the enzyme activity of the reaction carried out with 20 mM
benzonitrile as a substrate at 50°C.
Fig. 6. SEM of H. lutea immobilized in hybrid matrix containing 20% AHPS,
showing cell lysis and spore formation at the end of the productive phase (after
five reincubations). Bar= 10µm
The favorable effect of the addition of the AHPS was also
established. Unlike our previous experiments the increase of
the percentage of organic part in the hybrid sol-gel matrix up to
20% led to an increase in the nitrilase activity. This is probably
due to the type of the included organic material. Another
important factor is the pore size in the matrices. It decreases
with the increase of the organic constituent content. However
encapsulation in the nanocomposite materials containing
polysaccharides allows the enzymatic substrates to penetrate
from the external aqueous solution to the cells because of
its porosity. The cells are firmly immobilized and not easily
released when using sol-gel hybrid matrices (21). The addition
of 40% AHPS did not significantly affect the decrease of
enzyme activity and the values were almost similar.
Conclusions
The results from the XRD-analysis show that all the studied
hybrids are in amorphous state. The presence of a hybrid
nanostructure with well-defined nanounits and their aggregates,
formed by self-organizing processes, is clearly observed
by AFM studies. The surface morphology and structure of
nanobuilding blocks in each synthesized hybrid is different
and depends on its chemical composition. It has been proven
that the synthesized hybrid nanomaterials are successfully
applied as matrices for immobilization of bacterial and fungal
cells which retained the capability to synthesize the hydrolytic
enzymes nitrilase and α-galactosidase, respectively.
Acknowledgements
This work was supported by Grant NT 2-01 and NT 2-02 of the
National Fund for Scientific Research, Republic of Bulgaria.
1274
REFERENCES
1. Ademark P., Larsson M., Tjerneld F., Stalbrand H.
(2001) Enzyme Microb. Technol., 29, 441-448.
2. Aleksieva P., Nacheva L., Bratovanova E., Tchorbanov
B. (2007) Compt. Rend. Acad. Bulg. Sci., 60, 691-696.
3. Angelova B., Fernandes P., Spasova D., Mutafov S.,
Pinheiro H.M., Cabral J.M.S. (2006) Microscopy Res.
Technique, 69, 613-617.
4. Cantarella M., Spera A., Alfani F. (1998) J. Membr. Sci.,
147, 279-290.
5. Chernev G., Samuneva B., Djambaski P., Salvado
I.M.M., Fernandes M.H. (2006) CEJC, 4, 81-91.
6. Chernev G., Aleksieva P., Nacheva L., Kabaivanova
L., Samuneva B., Djambaski P., Miranda Salvado I.M.
(2007) XV international workshop on bioencapsulation,
Viena, Austria, 6-8 September, P1-15.
7. Dey P.M., Patel S., Brownleader M.D. (1993) Biotechnol.
Appl. Biochem., 17, 361-371.
8. Fawcett J.K., Scott J.E. (1960) J. Clin. Pathol., 13, 156-159.
9. Graham D., Pereira R., Barfield D., Don Covan (2000)
Enzyme Microb. Technol., 26, 368-373.
10.Kabaivanova L., Dobreva E., Emanuilova E., Chernev
G., Samuneva B., Salvado I. (2006) Minerva Biotechnol.,
18, 23-29.
11.Kickelbick G. (2003) Prog. Polym. Sci., 28, 83-114.
12.Kotwal S.M., Gote M.M., Sainkar S.R., Khan M.I.,
Khire J.M. (1998) Process Biochem., 33, 337-343.
13.Kotwal S.M., Gote M.M., Khan M.I., Khire J.M. (1999)
J. Ind. Microbiol. Biotechnol., 23, 661-667.
14.Luonteri E., Alatalo E., Siika-aho M., Penttila M.,
Tenkanen M. (1998) Biotechnol. Appl. Biochem., 28,
179-188.
15.Mulimani V.H., Ramalingam (1995) Biochem. Mol.
Biol. Int., 36, 897-905.
16.Naganagouda K., Prashanth S.J., Shankar S.K.,
Dhananjay S.K., Mulimani V.H. (2007) World J.
Microbiol. Biotechnol., 23, 1131-1137.
17.Padmakumar R., Oriel P. (1999) Appl. Biochem.
Biotechnol., 77, 671-680.
18.Pope E.J.A. (1995) J. Sol-Gel Sci. Tech., 4, 225-229.
19.Samuneva B., Kabaivanova L., Chernev G., Djambaski
P., Kashchieva E., Emanuilova E., Salvado I.M.M.,
Fernandes M.H.V., Wu A. (2008) J. Sol-Gel Sci. Technol.,
48, 73-79.
20.Schimidt H. (1994) J. Sol-Gel Sci. Tech., 1, 217-231.
21.Schipunov Y., Karpenko T., Bakunina I., Burtseva Y.
(2004) J. Biochem. Biophys. Methods, 59, 25-38.
22.Spasova D., Aleksieva P., Nacheva L., Kabaivanova L.,
Chernev G., Samuneva B. (2008) Z. Naturforsch., 63c,
893-897.
23.Wang M., Lu G., Ji G., Huang Z., Meth-Cohn O., Colby
J. (2000) Tetrahedron Assymetry, 11, 1123-1135.
Biotechnol. & Biotechnol. Eq. 23/2009/2